The surface coverage of micro-nano structure seriously affects the spectroscopic conformity of the surface-enhanced Raman scattering (SERS) substrate. In the paper, the influence of the surface coverage on the spectrum is studied, and a method is proposed to eliminate the influence of surface coverage. First, the SERS substrate was prepared by using the lyotropic liquid crystal soft template and the relationship between the surface coverage and Raman spectral intensity is analyzed to obtain a formula. Then, based on the this formula, all measured spectral intensities are unified to the standard surface coverage, which is defined with the measured average spectral intensity corresponded surface coverage, thus, the influence of the surface coverage on the spectral intensity is elimanated. Finally, the effectiveness of the proposed method is verified with different SERS substrates and different test spot sizes on the same substrate. The fitting and experimental results show that the spectral intensity changes as an exponential function of the surface coverage, the coefficients of variation are reduced to about 6% for different substrates, the spectroscopic conformities of the substrate are improved more than twice or so for different measured spots, and the coefficients of variation linearly decrease with the spots increase. In a word, the proposed method can improve the spectroscopic conformity of the SERS substrate significantly, the quantitative and reliable detection of various material components will be realized. This work may greatly promote the application of the SERS substrate in multiple detection fields.